Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors.

Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals.

Sedaxane-Use of Nuclear Receptor Transactivation Assays, Toxicogenomics, and Toxicokinetics as Part of a Mode of Action Framework for Rodent Liver Tumors.

Experimental data demonstrate a mode of action (MOA) for liver tumors in male rats and mice treated with sedaxane that starts with activation of CAR, followed by altered expression of CAR-responsive genes, increased cell proliferation, and eventually clonal expansion of preneoplastic cells, leading to the development of altered foci and tumors. This MOA is nonrelevant to human risk assessments. Methods and results in the MOA work for sedaxane illustrate promising directions that future MOA studies may be able to employ, in the spirit of "Tox21" and reduction of in vivo animal use: (1) currently available in vitro CAR and PXR reporter assays demonstrated that sedaxane is a direct CAR activator in mice and rats, and a weak PXR activator in rats; (2) mouse liver microarray results compared with a published CAR biomarker signature (based on 83 genes) showed a clear, statistical match, and a lack of correlation to similar biomarker signatures for AhR, PPARα, and STAT5B; (3) Ki67 immunohistochemistry and zonal image analysis showed significant increases in this marker of cell proliferation in mouse liver, without the need to dose a DNA labeling agent; and (4) toxicokinetic analysis of Cmax levels of sedaxane in blood showed a marked species difference between mice and rats that helps to explain differences in sensitivity to sedaxane. Incorporating these tools into the study plan for a new agrochemical or drug during development offers a promising alternative to the traditional need to conduct later, specialized MOA studies after the results of chronic bioassays are known.

Dermal absorption for pesticide health risk assessment: Harmonization of study design and data reporting for North American Regulatory submissions.

Although an internationally-adopted in vitro dermal absorption test guideline is available (OECD Test Guideline 428), the replacement of the in vivo approach in North America for pesticide formulations has not occurred due to concern over the reliability and consistency of the in vitro results. A 2012 workshop convened a panel of experts in the conduct of in vitro studies used for pesticide risk assessment, together with North American regulators, to examine techniques for in vitro dermal absorption testing. Discussions led to the recommended "best practices" for the conduct of in vitro dermal absorption studies provided herein. The workshop participants also developed recommendations for reporting study results in order to improve the quality and consistency of the data submitted to regulatory agencies in North America. Finally, a case study is presented that illustrates the use of the "triple-pack" approach; the studies, conducted for the registration of sulfoxaflor, follow the standardized recommendations provided at the workshop. In addressing the concerns of these regulators and of the regulated community, and providing harmonized recommendations to facilitate comparative data analyses, it is anticipated that wider acceptance of in vitro dermal absorption studies alone can be achieved for pesticide risk assessment.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

A predictive data-driven framework for endocrine prioritization: a triazole fungicide case study.

The US Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a tiered screening approach to determine the potential for a chemical to interact with estrogen, androgen, or thyroid hormone systems and/or perturb steroidogenesis. Use of high-throughput screening (HTS) to predict hazard and exposure is shifting the EDSP approach to (1) prioritization of chemicals for further screening; and (2) targeted use of EDSP Tier 1 assays to inform specific data needs. In this work, toxicology data for three triazole fungicides (triadimefon, propiconazole, and myclobutanil) were evaluated, including HTS results, EDSP Tier 1 screening (and other scientifically relevant information), and EPA guideline mammalian toxicology study data. The endocrine-related bioactivity predictions from HTS and information that satisfied the EDSP Tier 1 requirements were qualitatively concordant. Current limitations in the available HTS battery for thyroid and steroidogenesis pathways were mitigated by inclusion of guideline toxicology studies in this analysis. Similar margins (3-5 orders of magnitude) were observed between HTS-predicted human bioactivity and exposure values and between in vivo mammalian bioactivity and EPA chronic human exposure estimates for these products' registered uses. Combined HTS hazard and human exposure predictions suggest low priority for higher-tiered endocrine testing of these triazoles. Comparison with the mammalian toxicology database indicated that this HTS-based prioritization would have been protective for any potential in vivo effects that form the basis of current risk assessment for these chemicals. This example demonstrates an effective, human health protective roadmap for EDSP evaluation of pesticide active ingredients via prioritization using HTS and guideline toxicology information.

A critical review of neonicotinoid insecticides for developmental neurotoxicity.

A comprehensive review of published and previously unpublished studies was performed to evaluate the neonicotinoid insecticides for evidence of developmental neurotoxicity (DNT). These insecticides have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in insects, poor penetration of the mammalian blood-brain barrier, and low application rates. Nevertheless, examination of this issue is warranted, due to their insecticidal mode of action and potential exposure with agricultural and residential uses. This review identified in vitro, in vivo, and epidemiology studies in the literature and studies performed in rats in accordance with GLP standards and EPA guidelines with imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran, which are all the neonicotinoids currently registered in major markets. For the guideline-based studies, treatment was administered via the diet or gavage to primiparous female rats at three dose levels, plus a vehicle control (≥20/dose level), from gestation day 0 or 6 to lactation day 21. F1 males and females were evaluated using measures of motor activity, acoustic startle response, cognition, brain morphometry, and neuropathology. The principal effects in F1 animals were associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition. No common effects were identified among the neonicotinoids that were consistent with DNT or the neurodevelopmental effects associated with nicotine. Findings at high doses were associated with evidence of systemic toxicity, which indicates that these insecticides do not selectively affect the developing nervous system.

Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action.

Toxicogenomics (TGx) is employed frequently to investigate underlying molecular mechanisms of the compound of interest and, thus, has become an aid to mode of action determination. However, the results and interpretation of a TGx dataset are influenced by the experimental design and methods of analysis employed. This article describes an evaluation and reanalysis, by two independent laboratories, of previously published TGx mouse liver microarray data for a triazole fungicide, propiconazole (PPZ), and the anticonvulsant drug phenobarbital (PB). Propiconazole produced an increase incidence of liver tumors in male CD-1 mice only at a dose that exceeded the maximum tolerated dose (2500 ppm). Firstly, we illustrate how experimental design differences between two in vivo studies with PPZ and PB may impact the comparisons of TGx results. Secondly, we demonstrate that different researchers using different pathway analysis tools can come to different conclusions on specific mechanistic pathways, even when using the same datasets. Finally, despite these differences the results across three different analyses also show a striking degree of similarity observed for PPZ and PB treated livers when the expression data are viewed as major signaling pathways and cell processes affected. Additional studies described here show that the postulated key event of hepatocellular proliferation was observed in CD-1 mice for both PPZ and PB, and that PPZ is also a potent activator of the mouse CAR nuclear receptor. Thus, with regard to the events which are hallmarks of CAR-induced effects that are key events in the mode of action (MOA) of mouse liver carcinogenesis with PB, PPZ-induced tumors can be viewed as being promoted by a similar PB-like CAR-dependent MOA.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

Re-evaluation of the Big Blue® mouse assay of propiconazole suggests lack of mutagenicity.

Propiconazole (PPZ) is a conazole fungicide that is not mutagenic, clastogenic, or DNA damaging in standard in vitro and in vivo genetic toxicity tests for gene mutations, chromosome aberrations, DNA damage, and cell transformation. However, it was demonstrated to be a male mouse liver carcinogen when administered in food for 24 months only at a concentration of 2,500 ppm that exceeded the maximum tolerated dose based on increased mortality, decreased body weight gain, and the presence of liver necrosis. PPZ was subsequently tested for mutagenicity in the Big Blue® transgenic mouse assay at the 2,500 ppm dose, and the result was reported as positive by Ross et al. ([2009]: Mutagenesis 24:149-152). Subsets of the mutants from the control and PPZ-exposed groups were sequenced to determine the mutation spectra and a multivariate clustering analysis method purportedly substantiated the increase in mutant frequency with PPZ (Ross and Leavitt. [2010]: Mutagenesis 25:231-234). However, as reported here, the results of the analysis of the mutation spectra using a conventional method indicated no treatment-related differences in the spectra. In this article, we re-examine the Big Blue® mouse findings with PPZ and conclude that the compound does not act as a mutagen in vivo.

Multi-species analyses of direct activators of the constitutive androstane receptor.

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.

Mouse liver effects of cyproconazole, a triazole fungicide: role of the constitutive androstane receptor.

Cyproconazole, a triazole fungicide, causes hepatocellular adenomas and carcinomas in CD-1 mice at dose levels of 100 and 200 ppm. The constitutive androstane receptor (CAR) has been shown to play a significant role in the overall mode of action for several nongenotoxic rodent carcinogens such as phenobarbital. The liver effects of dietary cyproconazole or phenobarbital were investigated after 2, 7, or 14 days in male CD-1, C57BL/6J, and C3H/HeNClrBR mice. Cyproconazole produced similar, dose-responsive effects in all three strains of mice, and the response was similar to that of phenobarbital. Subsequently, Car-null and wild-type male mice on a C3H/HeNClrBR background were administered 200 or 450 ppm cyproconazole, or 850 ppm phenobarbital for up to 7 days. In wild-type mice, 200 ppm cyproconazole caused liver hypertrophy, increased liver weight and cell proliferation, single-cell necrosis and fat vacuolation, effects generally similar to those caused by 850 ppm phenobarbital. Plasma cholesterol was decreased by both compounds, but cyproconazole had a greater effect. The higher dose (450 ppm) of cyproconazole caused similar changes, but greater evidence of liver damage was observed, including a large increase in plasma transaminases. Induction of CAR target genes Cyp2b10 and Gadd45beta was observed with both compounds, whereas the cell cycle regulatory gene Mdm2 was unaffected. In Car-null mice, the effects noted with either cyproconazole or phenobarbital were absent or greatly diminished. These experiments demonstrate that short-term liver effects of cyproconazole in mice are CAR-dependent and similar to those of phenobarbital, a known nongenotoxic rodent liver carcinogen.

Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 2: species differences in response.

Thiamethoxam is a neonicotinoid insecticide that is not a mutagen, but it did cause a significant increase in liver cancer in mice, but not rats, in chronic dietary feeding studies. Previous studies in mice have characterized a carcinogenicity mode of action that involved depletion of plasma cholesterol, cell death, both as single cell necrosis and as apoptosis, and sustained increases in cell replication rates. In a study reported in this article, female rats have been exposed to thiamethoxam in their diet at concentrations of 0, 1000, and 3000 ppm for 50 weeks, a study design directly comparable to the mouse study in which the mode of action changes were characterized. In rats, thiamethoxam had no adverse effects on either the biochemistry or histopathology of the liver at any time point during the study. Cell replication rates were not increased, in fact they were significantly decreased at several time points. The lack of effect on the rat liver is entirely consistent with the lack of liver tumor formation in the two-year cancer bioassay. Comparisons of the metabolism of thiamethoxam in rats and mice have shown that concentrations of the parent chemical were either similar or higher in rat blood than in mouse blood in both single dose and the dietary studies strongly indicating that thiamethoxam itself is unlikely to play a role in the development of liver tumors. In contrast, the concentrations of the two metabolites, CGA265307 and CGA330050, shown to play a role in the development of liver damage in the mouse, were 140- (CGA265307) and 15- (CGA330050) fold lower in rats than in mice following either a single oral dose, or dietary administration of thiamethoxam for up to 50 weeks. Comparisons of the major metabolic pathways of thiamethoxam in vitro using mouse, rat, and human liver fractions have shown that metabolic rates in humans are lower than those in the rat suggesting that thiamethoxam is unlikely to pose a hazard to humans exposed to this chemical at the low concentrations found in the environment or during its use as an insecticide.

Case study: weight of evidence evaluation of the human health relevance of thiamethoxam-related mouse liver tumors.

Thiamethoxam (CGA293343; 3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine) was shown to increase the incidence of mouse liver tumors in an 18-month study; however, thiamethoxam was not hepatocarcinogenic in rats. Thiamethoxam is not genotoxic, and, given the late life generation of mouse liver tumors, suggests a time-related progression of key hepatic events that leads to the tumors. These key events were identified in a series of studies of up to 50 weeks that showed the time-dependent evolution of relatively mild liver dysfunction within 10 weeks of dosing, followed by frank signs of hepatotoxicity after 20 weeks, leading to cellular attrition and regenerative hyperplasia. A metabolite, CGA330050, was identified as generating the mild hepatic toxicity, and another metabolite, CGA265307, exacerbated the initial toxicity by inhibiting inducible nitric oxide synthase. This combination of metabolite-generated hepatotoxicity and increase in cell replication rates is postulated as the mode of action for thiamethoxam-related mouse liver tumors. The relevance of these mouse-specific tumors to human health was assessed by using the framework and decision logic developed by ILSI-RSI. The postulated mode of action was tested against the Hill criteria and found to fulfill the comprehensive requirements of strength, consistency, specificity, temporality, dose-response, and the collective criteria of being a plausible mode of action that fits with known and similar modes of action. Whereas the postulated mode of action could theoretically operate in human liver, quantitation of the key metabolites in vivo and in vitro showed that mice, but not rats or humans, generate sufficient amounts of these metabolites to initiate the hepatic toxicity and consequent tumors. Indeed, rats fed 3000 ppm thiamethoxam for a lifetime did not develop hepatotoxicity or tumors. In conclusion, the coherence and extent of the database clearly demonstrates the mode of action for mouse liver tumorigenesis and also allows for the conclusion that thiamethoxam does not pose a carcinogenic risk to humans.